The repair of alkylation damage to the genome is critical in all cells, because such damage is cytotoxic and potentially mutagenic. These repair pathways counteract endogenous and environmental alkylating agents. Alkylation chemotherapy is a major therapeutic modality for many tumors, underscoring the importance of these pathways in cancer. A number of different pathways exist for alkylation repair, which include base excision and nucleotide excision repair, direct reversal by methyl-guanine methyltransferase (MGMT), and dealkylation by the AlkB protein family. We recently demonstrated that one of the human AlkB homologues, ALKBH3, plays an important role in alkylation damage repair in specific tumor cells, but is dispensable for this function in nonmalignant cells. This suggests that alkylation repair enzymes may be differentially regulated in various cellular contexts. While much is known about the biochemical mechanisms that mediate DNA alkylation repair, we know very little about how these pathways are modulated, particularly in metazoans. We have recently discovered a critical role for the deubiquitinase OTUD4 in the regulation of MGMT and the AlkB family of enzymes in human cells. Our data suggests that OTUD4 functions as a component of a complex with multiple, differentially regulated deubiquitinase activities to control alkylation repair, as well as impacting DNA damage signaling. Together, our work implicates ubiquitination as a critical regulator of alkylation damage repair, an unexplored area which we seek to understand in this proposal. Specifically, we will test the hypothesis that OTUD4 serves as a deubiquitinase complex scaffold that promotes alkylation chemoresistance, both in vitro and in a mouse tumor model (Aim 1). We will also determine how the catalytic specificity of this deubiquitinase may be regulated by post- translational modification to modulate DNA damage response signaling (Aim 2). Finally, we will test the hypothesis that non-proteasomal ubiquitination plays a role in the activation of the ALKBH3 pathway (Aim 3). Beyond its implications on our understanding of DNA repair pathways, this work will provide new insights into tumor responses to alkylation chemotherapy, and may reveal novel small molecule targets for chemo sensitization.